My goal is to lead a translational research group focused on breast cancer prevention. The Career Development Award to Promote Diversity (K22) will allow me to gain additional research training in the areas of human breast pre-malignancy and to gather the critical preliminary data that will be the foundation of my future proposals as I pursue an independent research position. Upon receipt of this prestigious award, I anticipate securing a position within two years and pursuing independent funding such as an RO1. During the first year of the award period, I will focus on my training as a translational researcher by taking the Introduction to Clinical and Translational Cancer Research Course and by participating in the Breast Cancer Working group, a meeting designed to the bring bench scientists, clinical investigators, patient care personnel, and public health researchers together to determine collaborative research directions and to obtain a major goal - the eradication of breast cancer. To develop my leadership and management skills as I prepare to lead my own independent research group, I will participate in the Professional Development Series offered throughout the year. I will meet weekly with my mentors to discuss my research progress, potential funding opportunities, and my search for an independent position. During my second year I plan to pursue independent funding and apply for faculty positions. I anticipate having gathered sufficient data from the experiments proposed below to provide a solid foundation for my own research program. This, along with the funding already in place by the K22, should place me in a strong position as I begin applying for faculty positions. Ductal carcinoma in situ (DCIS) is a non-invasive cancer lesion that progresses to invasive cancer approximately half the time. The ability to predict DCIS malignant progression could significantly improve patient treatment and survival. However, we are not currently able to distinguish those DCIS lesions with invasion potential. Studies have identified a subpopulation of cells within breast cancer lesions which appear to drive metastasis and epithelial-mesenchymal transition (EMT). These tumor initiating or cancer stem cells share many of the properties of their normal stem cell counterparts such as long term self-renewal potential. Additionally, several groups recently demonstrated that ovarian steroid hormones indirectly influencing mammary stem cell numbers and cancer progression. The objective of this application is to characterize the steroid hormone paracrine signaling pathways involved in aberrant stem cell self-renewal and invasion which have potential for development of subtype-specific therapies. Our hypothesis is that the risk for DCIS malignant progression is determined by the aberrant self-renewal pathways in DCIS tumor initiating cell populations. In vitro assays of self-renewal and invasion potential, and a novel in vivo method recently developed by our laboratory, the MIND (mouse intraductal) model, will be utilized in this proposal to test our hypothesis. The MIND technique is a xenograft model of human DCIS in which primary human DCIS cells are engrafted intraductally into mammary glands of immunodeficient mice. This model mimics DCIS in vivo in the mouse to the closest condition that it is found in the human since DCIS initiates inside the ducts. Therefore the mammary ducts provide a natural microenvironment for DCIS cell growth. Specific Aim 1 is to identify and characterize the stem cell population's role in growth and invasion potential among DCIS subtypes in vivo and in vitro. Primary human cells of each DCIS subtype (Luminal A & B, basal, and HER2 over expressing) will be used to examine self-renewal ability, invasion potential, and the epithelial mesenchymal transition (EMT) phenotype of potential stem cell populations using both in vivo and in vitro techniques. Specific Aim 2 is to determine the influence of ER and PR paracrine signaling on primary human DCIS cells stem growth, self- renewal, and invasion potential using mammosphere assays and the MIND model. We will assess the paracrine effects of steroid hormone treatment on self-renewal ability, invasive progression, and EMT by in vitro mammospheres assays and in vivo MIND xenotransplantation. The molecular characterization of tumor initiating cells and the signaling pathways controlling their fate is promising to lead to the development of individualized therapeutic strategies for prevention of DCIS and/or invasive progression. My ultimate goal is to use our novel model of human primary DCIS, which mimics the heterogeneity of human non-invasive breast lesions, to study the mechanisms underlying progression to invasive breast cancer. In the long term, these studies should facilitate more accurate risk assessment and the design of therapeutic strategies for prevention based on a better understanding of the distinct mechanisms of malignant progression. The data obtain will be the foundation of my future grant proposals and completion of these proposed experiments is essential to the development of my own independent research program.